Our objective is to develop a detailed understanding of the molecular and structural basis for diverse functions of protein 4.1R and its homologs (4.1G, 4A B and 4.1N). Detailed molecular analysis revealed that 4.1R and its homologs are characterized by the presence of three highly conserved functional domains interspersed with domains unique to individual members of the 4.1 family. We hypothesize that the unique non-conserved domains in different 4.1 proteins could play an important role by regulating the function of the conserved functional domains and/or by enabling the individual 4.1 homologs to participate in novel, yet to be, [sic] defined protein interactions. Furthermore, we hypothesize that regulation of the diverse cellular functions by 4.1 involves 4.1-mediated assembly/disassembly of supramolecular protein complexes between membrane proteins, signaling molecules and cytoskeletal proteins and that the assembly/disassembly of the protein complexes is regulated by 4.1 phosphorylation and by the interaction of 4.1 with phospholipids. To achieve these objectives, we propose the following specific aims: 1) Explore the hypothesis that the non-conserved N-terminal domain of 4.1 plays a role in regulating the membrane binding function and determine the structural basis for this regulatory role. 2) Explore the function of the non-conserved C-terminal domain of 4.1R in differentiating epithelial cells. 3) Explore the effects of phosphorylation and interaction with anionic phospholipids on 4.1R function. Specifically, we will study the effects of these modifications on the ability of 4.1R to anchor the spectrin-based skeleton to the lipid bilayer and in regulating red cell membrane mechanical properties. 4) Explore the structural basis for the multiple functions of 4.1R by determining the crystal structure of 4.1R-membrane protein complexes and of the spectrin-actin binding and the conserved C-terminal domains of 4.1 R. We anticipate that the successful accomplishment of our research objectives will shed new insights into the structural basis for the assembly/disassembly of organelle and cell-specific supramolecular protein complexes. By focusing on previously unexplored aspects of 4.1 structure and function, we hope to significantly extend our understanding of how diverse isoforms of a cytoskeletal protein generated by alternative splicing can dynamically regulate multiple cellular functions.